micrornas role in hepatitis c virus pathogenesis

Accepted Manuscript MicroRNAs: Role in hepatitis C virus pathogenesis Shubham Shrivastava, Robert Steele, Ranjit Ray, Ratna B Ray PII: S2352-3042(15)00006-9 DOI: 10.1016/j.gendis.2015.01.001 Reference: GENDIS 36 To appear in: Genes & Diseases Received Date: December 2014 Accepted Date: January 2015 Please cite this article as: Shrivastava S, Steele R, Ray R, Ray RB, MicroRNAs: Role in hepatitis C virus pathogenesis, Genes & Diseases (2015), doi: 10.1016/j.gendis.2015.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT RI PT MicroRNAs: Role in Hepatitis C Virus pathogenesis Shubham Shrivastava a, Robert Steele a, Ranjit Ray b, Ratna B Ray a Departments of Pathology, Saint Louis University, St Louis, Missouri, USA b Departments of Internal Medicine, Saint Louis University, St Louis, Missouri ,USA M AN U SC a Running Title: miRNA in HCV infection EP TE D Key words: HCV, microRNA, liver disease, interferon signaling, circulatory miRNAs Requests for reprint: Ratna B Ray, Department of Pathology, Saint Louis University, AC C DRC 207, 1100 South Grand Boulevard, St Louis, MO 63104 Phone: 314-977-7822; Fax: 314-771-3816; E-mail: rayrb@slu.edu Abstract Hepatitis C virus (HCV) is a global health burden with an estimated 170-200 million peoples chronically infected worldwide HCV infection remains as an independent risk factor for chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, ACCEPTED MANUSCRIPT and a major reason for liver transplantation Discovery of direct acting antiviral (DAA) drugs have shown promising results with more than 90% success rate in clearing the HCV RNA in patients, although long-term consequences remain to be evaluated microRNAs (miRNAs) are important players in establishment of HCV infection and RI PT target crucial host cellular factors needed for productive HCV replication and augmented cell growth Altered expression of miRNAs is involved in the pathogenesis associated with HCV infection by controlling signaling pathways such as immune response, proliferation and apoptosis miRNA is emerging as a means of communication between SC various cell types inside the liver There is likely possibility of developing circulating miRNAs as biomarkers of disease progression and can also serve as diagnostic tool with M AN U potential of early therapeutic intervention in HCV associated end stage liver disease This review focuses on recent studies highlighting the contribution of miRNAs in HCV life AC C EP TE D cycle and their coordinated regulation in HCV mediated liver disease progression ACCEPTED MANUSCRIPT Introduction Hepatitis C virus (HCV) is a hepatotropic, enveloped, single stranded and positive RI PT sense RNA virus belongs to family flaviviridae and genus hepacivirus The viral genome contains 5’ and 3’ untranslated regions (UTR) that are important for viral replication and translation An internal ribosome entry site (IRES) directs the synthesis of a single precursor polyprotein of approximately 3010 amino acids, which is cleaved by viral and SC cellular proteases into three structural (core, E1 and E2) and seven non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) viral proteins Core protein forms the capsid, which is surrounded by a lipid bilayer containing the glycoproteins, E1 and E2.1 These M AN U viral proteins both singly or in coordinated manner interact with host cellular factors and regulate various signaling pathways to facilitate virus mediated persistent infection.2 HCV is a major cause of chronic liver disease, mostly asymptomatic in nature Majority of infected patients, approximately 80%, develop persistent chronic infection and are at high risk for liver cirrhosis and hepatocellular carcinoma (HCC) An estimated and about 2.7-3.9 TE D 170-200 million peoples worldwide are infected with hepatitis C million peoples are living with HCV infection in the United States.4 In addition, HCC and cirrhosis have been increasing among persons infected with HCV.5,6 Advances in anti-HCV therapy have moved into a new era with interferon (IFN)-free regimens EP Treatment options are evolving and the once difficult to treat genotype (genotype 1) has shown significant improvements in sustained virologic response (SVR) rates All-oral, AC C IFN-free combinations of drugs are expected to cure more than 90% of infections.7-9 Direct-acting antivirals (DAAs) target nonstructural proteins of HCV resulting in the termination of viral replication Several antiviral drugs targeting viral and host factors essential for productive HCV infection is depicted in Figure The first NS3/4A protease inhibitors boceprevir (Merck, Whitehouse Station, NJ) and telaprevir (Vertex Pharmaceuticals, Cambridge, MA) have shown improved rate of SVR in genotype HCV infected patients in compared with standard pegylated interferon (PEG-IFN) and ribavirin (RBV) therapy, but their toxicities combined with PEG-IFN and RBV limited their overall efficacy Simeprevir (Janssen Pharmaceuticals, Titusville, NJ), faldaprevir, ACCEPTED MANUSCRIPT and asunaprevir are second-wave, first-generation NS3/4A inhibitors that have already been or will soon be approved Second-generation protease inhibitors are in clinical trials Daclatasvir (BMS-790052, Bristol-Myers Squibb Company) is the first approved DAA belonging to the class of NS5A replication complex inhibitors The potency of daclatasvir RI PT is very high, and this drug is an important component of combination regimens for all genotypes Sofosbuvir, the first approved NS5B polymerase inhibitor (Gilead Sciences, Foster City, CA) has shown high potency and lower drug resistance in clinical trials DAA drugs as anti-HCV therapeutics is highly encouraging.7,8 However, the efficacy of SC these new therapeutic options for cirrhotic patients, the most-difficult-to-treat population and long-term follow-up data will be needed to confirm the excellent outcome of SVR M AN U and liver pathogenesis An alternative or complementary approach to treat HCV infection is by targeting host factors that support viral life cycle Identifying host factors have emerged as a promising alternative since they will have a high barrier for viral resistance and can be broadly effective against all HCV genotypes.10 Several host factors were identified by using high throughput gene silencing screening approaches that helps in HCV entry, replication, assembly and release The most promising host factors are cyclophilin A (cypA), scavenger receptor (SR)-BI and TE D miR-122, phosphatidylinositol-4-kinase III alpha Drugs targeting HCV entry factors, such as SR-BI may prevent the initiation of new infection Cyclophilins are important host factors required for viral replication and Cyp A has been shown to interact with EP NS5A.10,11 The liver specific miR-122 binds to the 5’ UTR of the HCV genome and helps in viral replication and enhances viral protein synthesis.12-15 Drugs targeting host factors AC C such as, small molecule inhibitor of SR-BI (ITX 5061) and cyclophilin inhibitors, alisporivir (Debio-025, Novartis) are in clinical trials 7,10,11 Anti-HCV therapeutics targeting miR-122 showed reduction in HCV viremia with no evidence of viral resistance and minimal side effects in chimpanzees chronically infected with hepatitis C.16 In addition, Miravirsen (a locked nucleic acid-modified antisense oligonucleotide for miR-122) showed prolonged dose-dependent reductions in HCV RNA levels in chronic HCV genotype infected patients in Phase IIa clinical trials by Santaris Pharma (Copenhagen, Denmark) 17 These alternative approaches targeting host factors in combination with current DAA drugs will help in strengthening the host’s innate ACCEPTED MANUSCRIPT immunity as well as interfere with host factors required for HCV induced pathogenesis RI PT MicroRNA biogenesis and its regulation MicroRNAs (miRNAs) were discovered in 1993 during a developmental timing experiment in the nematode Caenorhabditis elegans Currently, human miRNA family has expanded to 2588 mature miRNAs (miRBase v21.0; http://www.mirbase.org/) and in silico prediction estimates that approximately 60% of human mRNA could be targets of SC miRNA These miRNAs account for only 1% of the human genome miRNAs are highly conserved in nearly all organisms and constitute a class of non-coding RNAs, about M AN U 18-22 nucleotides long and play a crucial role in the regulation of gene expression.18,19 Genes encoding miRNAs are transcribed by RNA polymerase II and form transcripts as primary miRNAs (pri-miRNAs) pri-miRNAs are processed by ribonuclease Drosha to produce precursor miRNAs (pre-miRNAs) which is exported into the cytoplasm and cleaved by the ribonuclease Dicer to produce mature, single stranded miRNAs.19-21 Once synthesized, mature miRNA binds to two proteins, GW182 and Argonaute/EIF2C (AGO) TE D family proteins and forms a complex called miRNA induced silencing complex (miRISC) and mediate the target mRNA recognition (Figure 2) miRNA regulation takes place at multiple steps, including their transcription, their processing by Drosha and Dicer, their loading onto AGO proteins and miRNA turnover.20,21 miRNA transcription is controlled EP by RNA Pol II-associated transcription factors and epigenetic regulators Transcription factors, such as p53, MYC, ZEB1 and ZEB2, and myoblast determination protein AC C (MYOD1) positively or negatively regulate miRNA expression Epigenetic control, such as DNA methylation and histone modifications also contribute to miRNA gene regulation miRNA identify target mRNA through specific base-pairing interactions between the 5' end of miRNA and sites within coding region and UTRs especially 3' UTR of mRNAs The domain at the end of miRNAs that spans from nucleotide position to is crucial for target recognition and has been termed the 'miRNA seed' The downstream nucleotides of miRNA (particularly nucleotide and less importantly nucleotides 13–16) also contribute to base pairing with the targets miRNAs with almost identical sequences ACCEPTED MANUSCRIPT at their ends forms miRNA seed families and they share targets For example, miR-17, miR-20 and miR-106 belongs to the same family by sharing a common seed sequence and they target a common gene, such as, the cyclin-dependent kinase inhibitor 1A (CDKN1A; also known as p21) Moreover, 64% of the human miRNAs are part of RI PT multimember seed families and therefore, co-expression of seed-related miRNAs induces a stronger downregulation of their common targets.22 miRNA inhibits the target gene expression either by mRNA degradation or translational repression The incomplete complementary binding leads to repression of translation or deadenylation of the target mRNA SC mRNA, whereas a complete complementary binding leads to degradation of the target miRNA promotes mRNA cleavage by inducing deadenylation or suppresses M AN U protein synthesis by repressing the translation initiation at the cap recognition or inducing ribosomes to drop off prematurely.19-21,23 Paradoxically, miRNA can also activate gene expression by targeting gene regulatory sequences miR-10a interacts with the 5' UTR of mRNAs encoding ribosomal proteins to enhance their translation.24 A putative target site for miR-373 has been identified in the promoter of E-cadherin and miR-373 overexpresssion has been shown to induce E-cadherin expression in prostate cancer cell 25 In another report, miR-369-3 is shown to be involved in the recruitment of Ago TE D line and fragile X mental retardation related protein (FXR1) genes and enhances the translation of tumor necrosis factor (TNF) mRNA during cell cycle arrest.26 A combinatorial nature of miRNA regulation i.e., each miRNA regulates hundreds of EP different mRNAs allow miRNA to be a part of complex regulatory networks in controlling gene expression in almost every biological process including development, AC C immune response, aging, cell proliferation and apoptosis Many microRNA genes are located in chromosomal regions frequently involved in chromosomal alterations such as deletion or amplification during tumor development.27 Therefore, it is not surprising that dysregulation of miRNA networks have been associated with cancer progression Virus-host interactions also involve several regulatory steps to control gene expression and one of them is changes in cellular miRNA expression profiles Cellular miRNAs control protein expression that may influence cellular tropism of viruses, modulate viral infectivity, and play a crucial role in inducing appropriate antiviral immune responses 28 Several RNA viruses have evolved ACCEPTED MANUSCRIPT mechanisms to degrade, boost, or hijack cellular miRNAs to benefit the viral life cycle 29 HCV infection exerts a profound effect on the expression of cellular miRNAs.30 Some of the cellular miRNAs affect viral replication directly by binding to the viral genome or RI PT indirectly by targeting host factors Direct Interaction of cellular miRNAs to the HCV genome Recent studies have identified several miRNAs as key players in virus-host SC interactions, regulating virus replication and pathogenesis during HCV infection The role of miR-122 in HCV infection was first demonstrated by sequestration of endogenous directly to M AN U miR-122 that led to a substantial reduction in HCV RNA abundance.12 miR-122 binds UTR of the virus genome at two adjacent sites in association with Ago2 It forms an oligomeric complex in which one miR-122 molecule binds to the 5' UTR of HCV RNA with 3' overhanging nucleotides, masking the 5' terminal sequences from nucleolytic degradation, thereby promoting viral RNA stability and propagation of HCV genome.14,31 Furthermore, specific internal nucleotides as well as 3' terminal nucleotides TE D in miR-122 were absolutely required for maintaining HCV RNA abundance.31 Recent study also demonstrated that miR-122 protects HCV RNA from 5' decay by targeting 5' exonuclease Xrn1 32 HCV genome harbors two more miR-122 target sites, one in the variable region of the 3’UTR and other in the NS5B coding region However, miR-122 EP binding to NS5B and 3’UTR impairs HCV RNA replication and translation 33 Exogenous expression of miR-122 allows efficient HCV RNA replication and/or infectious virion AC C production in non-permissive cell line.34-36 Besides miR-122, other miRNAs such as, miR-448, miR-196, miR-199a, let-7b and miR-181c have been reported to interact directly with HCV genome however, upon binding to HCV RNA, they inhibit HCV replication (Figure 3) Overexpression of miR-448 and miR-196 were able to substantially attenuate viral replication by directly targeting CORE and NS5A coding region of the HCV genome, respectively 37 Overexpression of miR-199a inhibited HCV replication in cells bearing HCV-1b or -2a genome length replicon on binding to stem-loop II region of 5’UTR of HCV genome.38 let-7b was also reported to directly target HCV genome and elicits anti-HCV activity.39 Mutational analysis identified let-7b ACCEPTED MANUSCRIPT binding sites at the coding sequences of NS5B and 5'-UTR of HCV genome that were conserved among various HCV genotypes We have recently showed that miR-181c is a novel miRNA that binds to E1 and NS5A regions of HCV genome and overexpression of miR-181c reduces the viral replication.40 However, miR-181c binding efficiency to RI PT genotype 1a and genotype 2a specific HCV genome is different Thus, further work is needed to understand the association between different genotypes of HCV genome and SC miRNAs Cellular miRNAs that regulates HCV replication by targeting interferon signaling M AN U pathway HCV has developed several strategies to evade the IFN signaling pathway to facilitate its own replication.41 HCV evades type I IFN pathway by inducing miRNAs that regulate the expression of target genes involved in innate immune response to viral infections The direct correlation of cellular miRNA in regulating type IFN signaling pathway to promote HCV replication was demonstrated by our group We established TE D that HCV induces the expression of miR-130a to evade IFN response by targeting IFITM1 Our study demonstrated that miR-130a expression is upregulated in liver biopsy from HCV infected patients as well as in HCV infected hepatocytes in vitro.42 Knockdown of miR-130a enhances IFITM1 expression that possesses anti-HCV EP activity.43 During IFN treatment, IFITM1 accumulates at hepatic tight junctions in the liver of HCV-infected patients and then, interacts with HCV co-receptors, including AC C CD81 and occludin, to disrupt the process of viral entry.44 Similar observation of reduced HCV RNA copies was reported in anti-miR-130a transfected virus infected cells 45 Subsequently, miR-130a expression was correlated with genes involved in transforming growth factor beta (TGF-β) signaling pathway and found to be reduced in HCV infection and upregulated on IFN treatment.45 However, another study reported that overexpression of miR-130a inhibit HCV replication by restoring the expression of endogenous IFN-α and IFN-β and interferon stimulated genes, MxA, ISG15 and USP18 respectively in TLR3 and RIG-I deficient hepatocytes.46 Upregulated miR-21 suppressed MyD88 and IRAK1 expression in HCV infected hepatocytes, which subsequently repressed type I ACCEPTED MANUSCRIPT IFN effector gene expression and the type I IFN-mediated antiviral response, thereby promoting viral replication.47 In a separate study, upregulation of miR-758 expression was reported in HCV infected patients and shown to be negatively correlated with decrease in TLR3 and TLR7 expression levels and thereby, reduced IFNα and IFNβ RI PT production to impair innate immune response.48 Overexpression of miR-122 has also been associated with inhibition of IFN signaling pathway Silencing of miR-122 enhances IFN-induced ISRE activity, by decreasing expression of SOCS3 This decrease in SOCS3 level was also regulated by enhanced methylation at SOCS3 gene promoter, implicating SC additional mechanism of inhibition of HCV replication using antisense oligonucleotides of miR-122.49 Recently, SOCS1 and SOCS3 were identified as the targets of miR-221 and overexpression of miR-221 was shown to accelerate anti-HCV effect of IFN-α in M AN U HCV infected hepatocytes.50 IFN-α treatment also modulates HCV-specific miRNAs expression in hepatocytes miR-324-5p and miR-489 shown to be upregulated in the presence of IFN-α while differential expression of miR-30c and miR-130a were observed between HCV-infected Huh7.5 cells treated with or without IFN-α.45 miR-30 cluster targets SOCS1 and SOCS3 genes that act as negative regulators of cytokine signaling TE D Specifically, SOCS1 and SOCS3 inhibit JAK tyrosine kinase activity and STATs in the JAK-STAT signaling pathway suggesting that IFN-α induced miRNAs modulates gene expression in HCV infected hepatocytes.45 IFN-β treatment of Huh7 cells showed an upregulation of miR-142-3p and miR-128a, and these miRNAs were downregulated in EP HCV replicon-expressing cells.51 IFN-β induced miRNAs, in conjunction with downregulation of miR-122, was also studied to prevent HCV replication Introduction of AC C anti-miRs against miR-196, miR-296, miR-351, miR-431 and miR-448, with and without the inclusion of miR-122 mimic, attenuated the IFN-β mediated reduction of viral RNA by ~75%.37 In recent study, a set of 750 miRNAs expression profiles was generated in response to IFN-α and interleukin (IL)-28B treatment to hepatocytes Let-7b was shown to inhibit HCV replication and viral protein translation by targeting host factor insulin-like growth factor mRNA-binding protein (IGF2BP1) Furthermore, inhibition of let-7b attenuated the anti-HCV effects of IFN-α and IL-28B.52 We recently observed that miR-373 is upregulated during HCV infection and negatively regulated type I IFN signaling pathway by suppressing JAK1 and IRF9 (unpublished observation) Together, ACCEPTED MANUSCRIPT Shulla A, Randall G Hepatitis C virus-host interactions, replication, and viral assembly Curr Opin Virol Dec 2012;2(6):725-732 Gravitz L Introduction: a smouldering public-health crisis Nature Jun 2011;474(7350):S2-S4 RI PT Smith BD, Morgan RL, Beckett GA, et al Centers for Disease Control and Prevention Recommendations for the identification of chronic hepatitis C virus infection among persons born during 1945-1965 MMWR Recomm Rep Aug 17 2012;61(RR-4):1-32 Kanwal F, Hoang T, Kramer JR, et al Increasing prevalence of HCC and cirrhosis in with chronic hepatitis C virus infection 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MicroRNA Science Sep 2005;309(5740):1577-1581 13 Henke JI, Goergen D, Zheng J, et al microRNA-122 stimulates translation of hepatitis C virus RNA EMBO J Dec 17 2008;27(24):3300-3310 14 Roberts AP, Lewis AP, Jopling CL miR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components Nucleic Acids Res Sep 2011;39(17):7716-7729 ACCEPTED MANUSCRIPT 15 Jangra RK, Yi M, Lemon SM Regulation of hepatitis C virus translation and infectious virus production by the microRNA miR-122 J Virol Jul 2010;84(13):6615-6625 16 Lanford RE, Hildebrandt-Eriksen ES, Petri A, et al Therapeutic silencing of 2010;327(5962):198-201 RI PT microRNA-122 in primates with chronic hepatitis C virus infection Science Jan 17 Janssen HL, Reesink HW, Lawitz EJ, et al Treatment of HCV infection by targeting microRNA N Engl J Med May 2013;368(18):1685-1694 SC 18 Friedman RC, Farh KK, Burge CB, Bartel DP Most mammalian mRNAs are conserved targets of microRNAs Genome Res Jan 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Sci U S A Feb 2008;105(5):1608-1613 26 Vasudevan S, Tong Y, Steitz JA Switching from repression to activation: microRNAs can up-regulate translation Science Dec 21 2007;318(5858):1931-1934 27 Croce CM Causes and consequences of microRNA dysregulation in cancer Nat Rev Genet Oct 2009; 10(10): 704–714 ACCEPTED MANUSCRIPT 28 Swaminathan G, Martin-Garcia J, Navas-Martin S RNA viruses and microRNAs: challenging discoveries for the 21st century Physiol Genomics Nov 15 2013;45(22):1035-1048 29 Roberts AP, Lewis AP, Jopling CL The role of microRNAs in viral infection Prog RI PT Mol Biol Transl Sci 2011;102:101-139 30 Shrivastava S, Mukherjee A, Ray RB Hepatitis C virus infection, microRNA and liver disease progression World J Hepatol Sep 27 2013;5(9):479-486 31 Machlin ES, Sarnow P, Sagan SM Masking the 5' terminal nucleotides of the Natl Acad Sci U S A Feb 22 2011;108(8):3193-3198 SC hepatitis C virus genome by an unconventional microRNA-target RNA complex Proc M AN U 32 Li Y, Masaki T, Yamane D, McGivern DR, Lemon SM Competing and noncompeting activities of miR-122 and the 5' exonuclease Xrn1 in regulation of hepatitis C virus replication Proc Natl Acad Sci U S A Jan 29 2013;110(5):1881-1886 33 Nasheri N, Singaravelu R, Goodmurphy M, Lyn RK, Pezacki JP Competing roles of microRNA-122 recognition elements in hepatitis C virus RNA Virology Feb 20 2011;410(2):336-344 miR-122 support TE D 34 Narbus CM, Israelow B, Sourisseau M, et al HepG2 cells expressing microRNA the entire hepatitis C virus life cycle J Virol Nov 2011;85(22):12087-12092 35 Kambara H, Fukuhara T, Shiokawa M, et al Establishment of a novel permissive cell EP line for the propagation of hepatitis C virus by expression of microRNA miR122 J Virol Feb 2012;86(3):1382-1393 AC C 36 Fukuhara T, Kambara H, Shiokawa M, et al Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus J Virol Aug 2012;86(15):7918-7933 37 Pedersen IM, Cheng G, Wieland S, et al Interferon modulation of cellular microRNAs as an antiviral mechanism Nature Oct 18 2007;449(7164):919-922 38 Murakami Y, Aly HH, Tajima A, Inoue I, Shimotohno K Regulation of the hepatitis C virus genome replication by miR-199a* J Hepatol Mar 2009;50(3):453-460 39 Cheng JC, Yeh YJ, Tseng CP, et al Let-7b is a novel regulator of hepatitis C virus replication Cell Mol Life Sci Aug 2012;69(15):2621-2633 ACCEPTED MANUSCRIPT 40 Mukherjee A, Shrivastava S, Bhanja Chowdhury J, Ray R, Ray RB Transcriptional suppression of miR-181c by hepatitis C virus enhances homeobox A1 expression J Virol Jul 2014;88(14):7929-7940 41 Shrivastava S, Ray RB Hepatitis C virus infection, autophagy and innate immune RI PT response In: Hayat MA, ed Autophagy Cancer, Other Pathologies, Inflammation, Immunity, Infection and Aging Academic Press, Elsevier 2014; 3:164-190 42 Bhanja Chowdhury J, Shrivastava S, Steele R, Di Bisceglie AM, Ray R, Ray RB Hepatitis C Virus Infection Modulates Expression of Interferon Stimulatory Gene SC IFITM1 by Upregulating miR-130A J Virol Sep 2012;86(18):10221-10225 43 Raychoudhuri A, Shrivastava S, Steele R, Kim H, Ray R, Ray RB ISG56 and proteins inhibit hepatitis 2011;85(24):12881-12889 C virus replication J Virol Dec M AN U IFITM1 44 Wilkins C, Woodward J, Lau DT, et al IFITM1 is a tight junction protein that inhibits hepatitis C virus entry Hepatology Feb 2013;57(2):461-469 45 Zhang X, Daucher M, Armistead D, Russell R, Kottilil S MicroRNA expression profiling in HCV-infected human hepatoma cells identifies potential anti-viral targets TE D induced by interferon-α PLoS One 2013;8(2):e55733 46 Li S, Duan X, Li Y, Liu B, McGilvray I, Chen L MicroRNA-130a inhibits HCV replication by restoring the innate immune response J Viral Hepat Feb 2014;21(2):121-128 EP 47 Chen Y, Chen J, Wang H, et al HCV-induced miR-21 contributes to evasion of host immune system by targeting MyD88 and IRAK1 PLoS Pathog 2013;9(4):e1003248 AC C 48 Yang Q, Fu S, Wang J 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associated with the response to interferon treatment of chronic hepatitis C BMC Med Genomics Oct 22 2010;3:48 SC 54 Sarasin-Filipowicz M, Krol J, Markiewicz I, Heim MH, Filipowicz W Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to M AN U interferon therapy Nat Med Jan 2009;15(1):31-33 55 Estrabaud E, Lapalus M, Broët P, et al Reduction of microRNA 122 expression in IFNL3 CT/TT carriers and during progression of fibrosis in patients with chronic hepatitis C J Virol Jun 2014;88(11):6394-6402 56 Köberle V, Waidmann O, Kronenberger B, et al Serum microRNA-122 kinetics in patients with chronic hepatitis C virus infection during antiviral therapy J Viral Hepat TE D Aug 2013;20(8):530-535 57 Shirasaki T, Honda M, Shimakami T, et al MicroRNA-27a regulates lipid metabolism and inhibits hepatitis C virus replication in human hepatoma cells J Virol May 2013;87(9):5270-5286 EP 58 Elhelw DS, Mekky RY, El-Ekiaby N, et al Predictive prognostic role of miR-181a with discrepancy in the liver and serum of genotype hepatitis C virus patients Biomed AC C Rep Nov 2014;2(6):843-848 59 McFarland AP, Horner SM, Jarret A, et al The favorable IFNL3 genotype escapes mRNA decay mediated by AU-rich elements and hepatitis C virus-induced microRNAs Nat Immunol Jan 2014;15(1):72-79 60 Jiang M, Broering R, Trippler M, et al MicroRNA-155 controls Toll-like receptor 3and hepatitis C virus-induced immune responses in the liver J Viral Hepat Feb 2014;21(2):99-110 61 Bala S, Tilahun Y, Taha O, et al Increased microRNA-155 expression in the serum and peripheral monocytes in chronic HCV infection J Transl Med Jul 30 2012;10:151 ACCEPTED MANUSCRIPT 62 Hsi E, Huang CF, Dai CY, et al Peripheral blood mononuclear cells microRNA predicts treatment outcome of hepatitis C virus genotype infection Antiviral Res May 2014;105:135-142 63 Choi Y, Dienes HP, Krawczynski K Kinetics of miR-122 expression in the liver RI PT during acute HCV infection PLoS One Oct 2013;8(10):e76501 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K, Szymańska B, Omulecka A, Cwiklińska H, Piekarska A EP Hepatic HMOX1 expression positively correlates with Bach-1 and miR-122 in patients with HCV mono and HIV/HCV coinfection PLoS One Apr 21 2014;9(4):e95564 AC C 70 Peng X, Li Y, Walters KA, et al Computational identification of hepatitis C virus associated microRNA-mRNA regulatory modules in human livers BMC Genomics Aug 11 2009;10:373 71 Steuerwald NM, Parsons JC, Bennett K, Bates TC, Bonkovsky HL Parallel microRNA and mRNA expression profiling of (genotype 1b) human hepatoma cells expressing hepatitis C virus Liver Int Nov 2010;30(10):1490-1504 72 Liu X, Wang T, Wakita T, Yang W Systematic identification of microRNA and messenger RNA profiles in hepatitis C virus-infected human hepatoma cells Virology Mar 2010;398(1):57-67 ACCEPTED MANUSCRIPT 73 Schuppan D, Krebs A, Bauer M, Hahn EG Hepatitis C and liver fibrosis Cell Death Differ Jan 2003;10 Suppl 1:S59-67 74 Marquez RT, Bandyopadhyay S, Wendlandt EB, et al Correlation between 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CypA, cyclophilin A; miR, microRNA; OCLN, occludin; EP SR-B1, scavenger receptor class B member 1; LD, lipid droplets Figure miRNA biogenesis and regulation of gene expression MicroRNAs (miRNAs) AC C are small non-coding RNA synthesized from protein coding genes or introns with the help of RNA polymerase II (1) First, the miRNA gene transcribed to a primary long transcript with stem loop structure as pri-miRNA (2) This pri-miRNA is processed by RNase III family of enzymes, Drosha with the help of double stranded RNA binding protein, DGCR8 and produce small ~70-nucleotide precursor hairpin structure as precursor miRNA (pre-miRNA) (3) Pre-miRNA then transported to the cytoplasm with the help of exportin5 protein (4) Pre-miRNA was further cleaved by Dicer together with transactivation-responsive (TAR) RNA-binding protein TRBP, in the cytoplasm and generate a ~20-bp miRNA/miRNA* duplex Following processing, one strand of the ACCEPTED MANUSCRIPT miRNA/miRNA*duplex (the guide strand) is preferentially loaded into the miRNA-induced silencing complex (miRISC) containing argonaute (AGO2) and form mature miRNA (5) Then, the mature miRNA targets specific messenger RNA (mRNA) RI PT at the seed region that lead to either mRNA degradation or inhibition of protein synthesis Figure Cellular miRNAs targeting HCV genome The binding of two copies of miR-122 within the 5’ UTR of the HCV genome enhances viral replication and translation The binding of miR-199a at 5’UTR inhibits viral replication Let-7b interacts SC with viral genome at two different locations, 5’UTR and NS5B coding region miR-448 targets Core and miR-196 targets NS5A of the HCV genome miR-181c inhibits viral M AN U replication by targeting E1 and NS5A regions of viral genome Figure Schematic representation of HCV infection associated miRNAs in liver disease progression Modulated expression of miRNAs regulates the expression of genes involved in several signaling pathways such as, IFN signaling, hepatocyte growth, lipid AC C EP TE D metabolism, inflammation and fibrosis linked with HCV induced liver disease AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ... HCV co-receptors, including AC C CD81 and occludin, to disrupt the process of viral entry.44 Similar observation of reduced HCV RNA copies was reported in anti-miR-130a transfected virus infected... markedly increased in patients infected with HCV Overexpression of miR-155 promoted the cell proliferation through activation of β-catenin and a concomitant increase in cyclin D1, c- myc, and survivin... HCC Chronic HCV infection induced liver fibrosis is mediated by upregulation of TGF-β.73 TGF-β signaling activates hepatic stellate cells (HSCs) to induce extracellular matrix production In HCV-infected